Characterization of microglial cells and their response to stimulation in an organotypic retinal culture system

Abstract

An organotypic culture system of the early postnatal rat retina was developed to study microglial activation within a tissue environment. One day after tissue preparation, microglial cells of the ganglion cell/nerve fiber layer revealed features of activation. Cells acquired an ameboid morphology as revealed by Bandeiraea simplicifolia lectin staining. Proliferation-as revealed by Ki67 immunocytochemistry-resulted in higher cell densities. In the supernatant, tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and monocyte chemoattractant factor-1 (MCP-1) were detected by using specific enzyme-linked immunosorbent assay systems, activated microglia being the most likely source of their release. After 6 days in vitro (div), microglial cells regained their resting morphology, and cell counts returned to control levels. Concomitantly, the release activity decreased to undetectable levels. When slices were treated at this later stage of cultivation (>6 div) with bacterial lipopolysaccharide (LPS; 100 ng/ml for 24 hours), microglial cells became activated, as revealed by a change in morphology. In parallel, the LPS treatment also resulted in high levels of TNF-alpha, IL-6, and MCP-1 in the culture medium. Both the release from the tissue and the morphological changes of the microglia were reversible. Seventy-two hours after LPS removal, only microglia with ramified morphology were found, and release activities returned to baseline. These data suggest that the organotypic culture of the retina is a useful model for studying microglial activation from its resting form.